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About this book

This completely revised and updated edition of YAC Protocols takes advantage of the many new advances in biology since the publication of the first edition, including the sequencing of the human genome, the genomes of a wide variety of other organisms, and the increased use of transgenic animals for understanding the molecular basis of human and animal disease. This volume also provides a much-needed update on the new techniques currently employed and to help redefine and illustrate the important roles still to be played by yeast artificial chromosome (YAC) technologies in the postgenomic age.

YAC Protocols, Second Edition, demonstrates the enormous potential use of YAC technologies in understanding the role of genetics in maintaining health, promoting pathogenesis, and conferring susceptibility to disease. Offering solutions to the many problems that have plagued researchers, this new edition allows for the wider and more efficient use of YAC technologies for the analysis of genetic and pathogenic causes of human disease.

Contents

Generation of Large Insert YAC Libraries. YAC Library Storage and Transport. YAC Library Screening I: Preparation of Hybridization Filters and PCR Pools. YAC Library Screening II: Hybridization and PCR-Based Screening Protocols. Cloning of Human Telomeres in Saccharomyces cerevisiae. Purification of YAC-Containing Total Yeast DNA. Restriction Analysis of YACs. RARE-Cleavage Analysis of YACs. YAC Localization by Fluorescence In Situ Hybridization. Alu-PCR Fingerprinting of YACs. Detection of Chimerism in YAC Clones. Amplification with Arbitrary Primers. End Rescue from YACs Using the Vectorette. Isolation of YAC Ends by Plasmid Rescue. End-Rescue of YAC Clone Inserts by Inverse PCR. Covering YAC-Cloned DNA with Phages and Cosmids. Fragmentation and Integrative Modification of YACs. Targeting Mutations to YACs by Homologous Recombination. Reconstruction of Large Genomic Segments of DNA by Meiotic Recombination Between YACs. Genomic Reconstruction by Mitotic Recombination of YACs. Amplification of the Copy Number of YACs. Transfer of YAC Clones to New Yeast Hosts. Use of ACEDB as a Database for YAC Library Data Management. YAC Transfer into Mammalian Cells by Cell Fusion. YAC Transfer by Microinjection. Transfection of Mammalian Cells via Lipofection. The Isolation of cDNAs by Hybridization of YACs to cDNA Libraries. cDNA Selection with YACs. Markers Selection and Media in YAC Cloning. Index.